Trafficking and localization studies of recombinant alpha1, 3-fucosyltransferase VI stably expressed in CHO cells.
Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3-fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.
Borsig
L
L
Institute of Physiology, University of Zürich, Switzerland.
Katopodis
A G
AG
Bowen
B R
BR
Berger
E G
EG
D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D005647:Fucosyltransferases / Q000737:chemistry* / Q000276:immunology; D006056:Golgi Apparatus / Q000201:enzymology; D006596:Hexosaminidases / Q000378:metabolism; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011994:Recombinant Proteins / Q000032:analysis; D014162:Transfection / Q000235:genetics
D007527:Isoenzymes; D011994:Recombinant Proteins; D005647:Fucosyltransferases; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase